Cloudman melanoma cells in culture have been used extensively for studies on hormonal regulation of pigmentation and proliferation. These functions are regulated by both melanotropin (MSH) and insulin. Specific, high-affinity binding sites for these hormones have been identified on the cells. A panel of mutant cell lines has been constructed with variant responses to MSH and/or insulin and a genetic hybridization system has been established for studying dominant/recessive relationships and identifying complementation groups involved in the various regulatory processes. Using an approach which integrates genetic and biochemical techniques, we have shown that MSH and insulin, though acting through different mechanisms, affect common or overlapping biochemical pathways. Apparently the pathways are regulated through the phosphorylation and dephosphorylation of specefic proteins. As many as eight phosphoproteins are regulated by MSH and as many as five phosphoproteins are regulated by insulin. The Beta subunit of the insulin receptor is one of the latter proteins. Another protein, pp90, is apparently a key regulatory molecule in the response of the cells to insulin. This protein is not detected in insulin-resistant mutants but is detected in complementing, insulin-sensitive hybrids between the mutants. The long-term objectives of this project are to determine the number of genes and the nature of the gene products involved in the response of Cloudman melanoma cells to MSH and to insulin. Specific aims include detailed genetic analyses of the responses to both MSH and insulin; identification of protein phosphorylation/dephosphorylation reactions regulated by MSH and by insulin; and investigations of the structure and function of receptors for both MSH and insulin. Studies on the receptors will focus in part on their potential roles as hormone-sensitive protein kinases. In all cases, wild-type, mutant, and hybrid cell lines will be analyzed to correlate specific biochemical events with specific cellular phenotypes. The major significance of the project is that a model system has been constructed for studying the action of two peptide hornomes from their initial cellular contact to their final regulation of the phenotypic state of the cell. The combined genetic and biochemical approach provides a powerful tool for conducting the study. The initial results have already proven useful to studies of other hormonal systems, as well as provided new information on the growth and differentiation of mammalian melanoma cells.